Poly ADP-ribose (pADP-R) may modify the rate of division of many cell types. The proliferative rate of cardiac cells in culture varies inversely with both ambient O2 and pADP-R concentration. We recently extended these original observations to skeletal muscle cells cultured in different O2 concentrations to assess the relationships between pADP-R, O2, and differentiation. After a period of rapid division, mononucleated myoblasts fuse to form differentiated, multinucleated myotubes. Muscle cells from 13 day chick embryo grown in 20% O2 form longer, wider myotubes with greater ramifications than do those grown in 5% O2. In these more highly differentaiting cultures activity of pADP-R polymerase, the enzyme responsible for pADP-R formation, is significantly higher at 72 hours than in cells grown in 5% 02 (P 0.02). When myotubes from cells grown in 20% 02 were separated from single cells, pADP-R polymerase activity was three times higher in myotubes than in single cells (P 0.02). The kinetic characteristics of the myotube and single cell pADP-R polymerase also differed significantly (Km for myotube =20 microns and for single cells 203 microns). These results suggest that pADP-R participates significantly in differentiation of skeletal muscle.